The level of production of a protein in a host cell is governed by three major factors: the number of copies of its gene within the cell, the efficiency with which those gene copies are transcribed and the efficiency with which the resultant messenger RNA ("mRNA") is translated. Efficiency of transcription and translation (which together comprise expression) is in turn dependent upon the nucleotide sequences which are normally situated ahead of the desired coding sequence or gene. These nucleotide sequences or expression control sequences define, inter alia, the location at which RNA polymerase interacts (the promoter sequence) to initiate transcription and at which ribosomes bind and interact with the mRNA (the product of transcription) to initiate translation.
Not all such expression control sequences function with equal efficiency. It is thus often of advantage to separate the specific coding sequence or gene for a desired protein from its adjacent nucleotide sequences and to fuse it instead to other expression control sequences so as to favor higher levels of expression. This having been achieved, the newly-engineered DNA fragment may be inserted into a higher copy number plasmid or a bacteriophage derivative in order to increase the number of gene copies within the cell and thereby further to improve the yield of expressed protein.
Because over-production of even normally non-toxic gene products may be harmful to host cells and lead to decreased stability of particular hostvector systems, an expression control sequence, in addition to improving the efficiency of transcription and translation of cloned genes, is also often made controllable so as to modulate expression during bacterial growth. For example, controllable expression control sequences are ones that may be switched off to enable the host cells to propagate without excessive build-up of gene products and then switched on to promote the expression of large amounts of the desired protein products, which are under the control of those expression control sequences.
Several expression control sequences, which satisfy some of the criteria set forth above, have been employed to express DNA sequences and genes coding for proteins and polypeptides in bacterial hosts. These include, for example, the operator, promoter and ribosome binding and interaction sequences of the lactose operon of E. coli (e.g., K. Itakura et al., "Expression In Escherichia coli Of A Chemically Synthesized Gene For The Hormone Somatostatin", Science, 198, pp. 1056-63 (1977); D. V. Goeddel et al., "Expression In Escherichia coli Of Chemically Synthesized Genes For Human Insulin", Proc. Natl. Acad. Sci. USA, 76, pp. 106-10 (1979)), the corresponding sequences of the tryptophan synthetase system of E. coli (J. S. Emtage et al., "Influenza Antigenic Determinants Are Expressed From Haemagglutinin Genes Cloned In Escherichia coli", Nature, 283, pp. 171-74 (1980); J. A. Martial et al., "Human Growth Hormone: Complementary DNA Cloning And Expression In Bacteria", Science, 205, pp. 602-06 (1979)) and the major operator and promoter regions of phage .lambda. (H. Bernard et al., "Construction Of Plasmid Cloning Vehicles That Promote Gene Expression From The Bacteriophage Lambda P.sub.L Promoter", Gene, 5, pp. 59-76 (1979); European patent application No. 41767).
Promoters for primer RNA and RNA I from the colicin EI genome (Col EI) are also known (E. M. Wong et al., "Temperature-Sensitive Copy Number Mutants Of Col EI Are Located In An Untranslated Region Of The Plasmid Genome", Proc. Natl. Acad. Sci. USA, 79, pp. 3570-74 (June 1982)). Among the group of promoters useful in the expression vectors and methods of this invention are these two promoters, i.e., the promoter for primer RNA (hereinafter designated "P.sub.m ") and the promoter for RNA I (hereinafter designated "P.sub.I "). A temperature sensitive copy number mutant of promoter P.sub.I is also known (E. M. Wong et al., supra). This promoter is also among those useful in this invention.
Promoters P.sub.I and P.sub.m are thought to be constitutive, i.e., they are not under the control of repressors, so that they continually promote expression of genes operatively-linked to them. Moreover, promoter P.sub.m may apparently be strengthened by mutation (E. M. Wong et al., supra).